Medulloblastoma, the most common type of pediatric brain malignancy, is an aggressive primitive neuroectodermal tumor arising from the cerebellum. It presents a significant cause of cancer-related death in children and current treatment of radio-chemotherapy impairs children's development and causes long-term adverse effects. Therefore, targeted therapies based on the molecular features of medulloblastoma are needed to minimize the treatment-related side effects in children. The transcription factor OTX2 is overexpressed and/or genomically amplified in most medulloblastomas. It is overexpressed in over 60% of medulloblastoma. Our previous study has identified OTX2 as a transcriptional repressor and a gatekeeper of myogenic and neuronal differentiation in medulloblastoma cells. The strong repressor activity exhibited by its homeobox domain (HD) renders OTX2 transcriptionally repressive. In medullomyoblastoma, a subtype of medulloblastoma with differentiated myogenic cells mixed with tumor cells, we have linked the loss of expression of OTX2 to the myogenic differentiation in tumor cell populations. Inducible knockdown of OTX2 by shRNA led to significant extension of survival in an orthotopic medulloblastoma xenograft model. Owing to the remarkable repressor activity displayed by the 60 amino acid OTX2-HD and its lack of functional motif related to transcriptional regulators, we hypothesize that this transcriptional repression is the result of interactions with epigenetic regulators. We therefore propose to test inhibitors of potential epigenetic regulators to characterize the nature of OTX2-HD mediated repression. We propose to perform in vivo crosslinking/immuno-purification/tandem mass spectrometry to identify binding partners of OTX2-HD. As a complementary strategy, we suggest additionally to use split-ubiquitin yeast two-hybrid system to identify novel interacting partners of OTX2-HD in a D425 medulloblastoma cDNA library. Subsequently, we will analyze and validate the interacting proteins in vitro and in vivo in medulloblastoma cells, and study the functionality of those binding proteins. In summary, understanding the molecular mechanism of OTX2 will advance our knowledge to develop targeted therapies that initiate the differentiation suppressed by OTX2 in medulloblastoma. This goal can be achieved by identifying the inhibitors of epigenetic regulators associated with OTX2- HD and by manipulating the potential interacting partners relevant in OTX2-mediated suppression of differentiation, which is the long term purpose of the study.